Enhancement of Acarbose Production in Actinoplanes sp. QQ-12 via Multiple Engineering Strategies

The α-glucosidase inhibitor acarbose is used in the treatment of type 2 diabetes mellitus. Metabolic engineering is crucial to overcome acarbose production bottlenecks. Herein, a genetic toolkit was developed to enable metabolic engineering in Actinoplanes sp.. The attachment/integration (Att/Int) systems of ΦBT1, pSAM2, R4, and TG1 showed conjugation frequencies of 0.98-24.4%. Furthermore, three mutants were constructed by deleting nontarget biosynthetic gene clusters (BGCs) and inserting one to three additional copies of the acarbose biosynthetic gene cluster (acb). These mutants, with 2, 3, and 4 copies of acb gene cluster, demonstrated titer increases of 69.4%, 99.3%, and 24.2%, respectively, with a sharply declined titer in the four-copy acb strain LGQ-17::3acb. To rescue the acarbose titer in LGQ-17::3acb, we overexpressed rate-limiting genes acbC, M, O, N, J, V, I, or S. The overexpression of acbJ and acbS resulted in acarbose titer increases of 1.04-fold and 98%, respectively. Furthermore, following fed-batch fermentation optimization in shake flasks, the titer of acarbose in LGQ-17::3acb::stnYp-acbJ increased by 1.1-fold to reach 8.12 g/L. This genetic engineering toolkit with multiple Att/Int systems and high conjugation frequencies paves the way for future genetic engineering in Actinoplanes sp., and the engineered strain shows excellent potential for industrial application.